Rational engineering of the longest transmembrane β barrel

Thesis

β-Barrel proteins, characterised by their closed cylindrical structures formed from β sheets, play crucial roles in both membrane-bound and soluble forms across a variety of biological processes. Specifically, multimeric transmembrane β barrels assemble from soluble monomers that undergo substantial conformational rearrangements to form mature pore structures. Though less common than single-chain β-barrel structures, this versatile protein fold underpins a wide array of applications in biotechnology, particularly as single-molecule sensors. While de novo designed sequences produce variations in β-strand number—affecting pore diameter—the ability to extend barrel length remains largely uncharted and poses challenges for predictive design. In this thesis, I report a β barrel that is 14.2 nm in length with a transmembrane domain—the longest of its kind, which was engineered by extending the heptameric α-hemolysin (αHL) pore with de novo designed sequences. The natural assembly mechanism is remarkably tolerant to elongation and drives correct barrel folding as confirmed by single-channel conductance measurements, chemically-detected alternating side-chain orientation, and TEM imaging. Barrel extension markedly enhances β-cyclodextrin retention at an internal binding site within the pore: the dwell time of β-cyclodextrin is ~70-fold longer in a barrel, which has been extended ~3-fold in length. The extension of the β barrel has no apparent limit and thereby unlocks untapped potential in sensor engineering.

Authors: He, H

• DOI: 10.5287/ora-key8pwqn6
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: nanopore; sensing; protein engineering
• Subjects: Chemical biology; Chemistry; Nanotechnology